Tannerella forsythia belongs to the ?red complex? of oral bacteria strongly associated with progression of chronic periodontitis. However, in contrast to Porphyromonas gingivalis and Treponema denticola (other members of the red complex), relatively little is known about virulence factors produced by T. forsythia. A feature in common between these and other periodontal pathogens is the production of high levels of proteolytic enzymes. In T. forsythia, the majority of proteolytic activity is due to six secreted proteases of unique subdomain structure (KLIKK proteases) belonging to five different families of serine and metallo- proteases. On the genomic level, every gene of the KLIKK protease is directly preceded by a gene encoding a putative lipoprotein in a seemingly co-transcriptional unit. Based on preliminary data, it is clear that at least two of these proteins are inhibitors of the adjacent protease. Therefore, we collectively designated these genes as FLIPPINs (Forsythia Lipoprotein Protease INhibitors). Flippins are small proteins (118-152 residues) which exhibit no sequence similarity with each other or with any other protein (the only exception is Flippins B1 and B2, which are 82% identical). Therefore, we hypothesize that Flippins are outer-membrane anchored protease inhibitors with novel structure and function, protecting T. forsythia from its own enzymes and thus essential for T. forsythia biological fitness. Furthermore, we theorize that they will also inhibit host proteases, as well as proteases secreted by other bacteria in the pathogenic subgingival biofilm to protect the native organism. Hence, this research proposal will investigate the important and innovative concept that, Flippins, through their ability to control proteolysis in the biofilm, may represent a new and previously unrecognized class of virulence determinants or modifiers. Therefore, the main objective of this proposal is to characterize function, expression, specificity and role in T. forsythia biology of selected Flippins as outlined in two Specific Aims: (i) characterization of the specificity spectrum and mechanism of protease inhibition by Flippins; and (ii) determination the regulation of selective Flippins expression, subcellular localization and their role in T. forsythia biology. The significance and impact of this project is that the functional characterization of Flippins may highlight novel regulatory mechanisms to control proteolytic activity and provide new insights on T. forsythia physiology, its role in the subgingival biofilm and mechanism of virulence. This knowledge will create novel perspectives in the treatment of periodontitis.